Download Development and Characterization of a LONG©R3 IGF

Company Information
Development and Characterization of a LONG©R3 IGF‐1 ELISA Repligen Corporation 41 Seyon Street Building #1, Suite 100 Waltham, MA 02453 www.repligen.com/bioprocessing
Ariane Marolewski*, Cathy Rich, Nipa Purohit
Repligen Corporation, Waltham, MA, USA
Accuracy, Precision, and Dilutional Linearity
Abstract
LONG®R3 IGF‐1 is a human IGF‐1 analog containing a 13 amino acid N‐terminal extension and a mutation at position 3. It activates the Type 1 IGF receptor, which is responsible for increasing cell growth and protein synthesis effects in CHO cells. A common growth factor supplement used in CHO media, insulin, also acts primarily through the IGF‐R. Since LONG®R3 IGF‐1 is manufactured under GMP and is used at low concentrations (10 – 100 ng/mL) in cell culture it represents a regulatory‐friendly method of enhancing serum‐free cell culture performance. Quantitation of LONG®R3 IGF‐1 is important when developing cell culture processes in order to optimize the concentration of LONG®R3 IGF‐1 used and feeding strategies. Quantitation is also important when developing a purification process, in order to demonstrate clearance. An ELISA has been developed for the quantitation of LONG®R3 IGF‐1 in media and drug substance samples. The assay uses a sandwich ELISA format with colorimetric detection. The performance characteristics of the assay are presented including accuracy, precision, linearity, LOQ, recovery from media, and range.
Schematic of LONG®R3IGF‐1 ELISA
Nominal Conc
(ng/mL)
Composition
Assays
Recovery
(Mean ± SD)
20
2.5
1.25
20
2.5
1.25
Diluent
Diluent
Diluent
Conditioned media
Conditioned media
Conditioned media
8
8
8
1
1
1
21.1 ± 1.9
2.5 ± 0.4
1.2 ± 0.2
20.4
2.2
0.9
For Dilutional Linearity, a sample was prepared at 80 ng/mL then 2‐fold serially diluted down to 5 ng/mL. Dilutions of 40, 20, 10, and 5 ng/mL were run in the ELISA. Recoveries were within 70‐130%. Specificity
Specificity of the ELISA was measured by testing responses to the related
molecules insulin and IGF‐1. Results showed that the antibodies have no reactivity
to insulin and only 35‐40% reactivity with IGF‐1.
LONG®R3IGF‐1 ELISA Instructions
Preparation of Standards (40 – 0.31 ng/mL)
Limits of Detection and Quantitation
The limit of quantitation is defined as the lowest concentration with recovery of 70‐130% of nominal. The LOQ for the ELISA is 0.63 ng/mL. Assay Instructions
Wash plate before use twice with 1x PBS
Add 100 µL/well Standards, Samples, and QC Control (as applicable) Incubate for 2 hours at 2‐8°C
Wash three times with PBS‐0.05% Tween 20
Add 100 µL/well Detection Antibody to the plate
Incubate for 1 hour at 2‐8°C
Wash three times with PBS‐0.05% Tween 20
Add 100 µL/well Streptavidin‐HRP to the plate
Incubate for 30 minutes at 2‐8°C
Wash two times with PBS‐0.05% Tween 20
Wash once with 1x PBS
Add 100 µL/well TM Blue to the plate
Develop then Stop with 100 µL/well 4 N Sulfuric Acid
Read plate at 450 nm
The limit of detection was defined as the lowest concentration with a signal/noise ratio greater than 3. Signal/noise ratio was determined as the absorbance of the highest standard divided by the blank. The LOD for the ELISA is 0.31 ng/mL.
Conclusions
•Due to LONG®R3IGF‐1’s ability to increase cell growth and productivity, it is a useful supplement in manufacturing processes. •In order to efficiently develop upstream and downstream processes it would be useful to quantitiate LONG®R3IGF‐1 levels in culture and during purification. •An ELISA kit has been developed that specifically, accurately, and precisely quantitates LONG®R3IGF‐1.
•LONG®R3IGF1is produced in a validated, cGMP (ICH Q7) compliant, manufacturing process with no animal derived components; Final product is released by QC testing
•Ask us about applications for LONG®R3IGF‐1 or visit [email protected]
•CHO culture: cell line development, cell banking
•Stem cell culture and regenerative medicine